Hydrogel microencapsulation of cells is a promising strategy for immunoprotection after transplantation. Since the development of alginate-poly-L-lysine encapsulation by Lim and Sun in 1980 (Lim, et al., Science 210(4472):908-10 (1980)), their approach has remained the standard for cell encapsulation, although major efforts have led to improvements (T. Wang, et al., Nature Biotechnology, 15:358 (1997)). The ease of alginate microencapsulation, along with alginate's inherent biotolerance in vivo, has led to its prevalence (E. Santos, et al., J. Control, 170:1 (2013)), even though the ability to control local cellular environment via incorporation of bioactive molecules (e.g., adhesive peptides) is limited. Highly tunable, synthetic hydrogel encapsulation is attractive for various regenerative medicine applications (Lutolf, et al, Nat Biotechnol, 23(1):47-55 (2005), Peppas, et al., Science, 263(5154):1715-20 (1994); Langer, et al., Nature, 428(6982):487-92 (2004)), not only for immunoisolation, but also for directing cell behavior and fate (Lutolf, et al, Nat Biotechnol, 23(1):47-55 (2005)). Several groups have developed more complex encapsulation configurations, such as cell encapsulation in natural hydrogel fibers (Onoe, et al., Nature Materials, 12(6):584-90 (2013); Jun, et al., Biomaterials, 34(33):8122-30 (2013)), but the benefits of added geometric complexity remain to be established.
Minimization of encapsulation volume is also important in many regenerative medicine scenarios, including pancreatic islet transplantation. In an effort to reduce the high polydispersity present in electrostatically generated alginate droplets with diameters <200 μm (Goosen, et al., J. Microencapsul., 13(5):497-508 (1997)), microfluidic droplet generation has been explored (Choi, et al., Biomed Microdevices, 9(6):855-62 (2007); Tan, et al., Advanced Materials, 19:2696 (2007); Um, et al., Microfluid Nanofluid, 5:541 (2008)). Microfluidic devices have also been used to generate synthetic hydrogel particles (Rossow, et al., Journal of the American Chemical Society, 134(10):4983-9 (2012), Velasco, et al., Small, 8(11):1633-42 (2012), Allazetta, et al., Biomacromolecules, 14(4):1122-31 (2013), Panda, et al., Lab Chip, 8(7):1056-61 (2008), Chung, et al., Applied Physics Letters, 91:041106 (2007)). Weitz established encapsulation of cells inside emulsions for high throughput cell-based assays Koster, et al., Lab Chip, 8(7):1110-5 (2008). However, translating this work into covalently crosslinking of microgels within microfluidic devices adds significant complexity because polymer precursors must be liquid while flowing through the focusing nozzle, but droplets must crosslink rapidly after being generated to prevent them from merging.
Synthetic polymer microgels have been generated, including cell-laden microgels (Rossow, et al., Journal of the American Chemical Society, 134(10):4983-9 (2012), Velasco, et al., Small, 8(11):1633-42 (2012), Allazetta, et al., Biomacromolecules, 14(4):1122-31 (2013), Panda, et al., Lab Chip, 8(7):1056-61 (2008), Chung, et al., Applied Physics Letters, 91:041106 (2007) Kesselman, et al., Small, 8(7):1092-8 (2012), Tumarkin, et al., Chemical Society Reviews, 38(8):2161-8 (2009)). However, even for synthetic polymer encapsulation, control of cellular microenvironment by functionalization of polymers with bioactive molecules remains a significant challenge. Most of these schemes require crosslinking using UV-based free radical polymerization, resulting in potentially cytotoxic effects on encapsulated cells. Although cell encapsulation in synthetic microgels crosslinked without free radicals has been reported, the polymer cannot easily be functionalized with bioactive molecules (Rossow, et al., Journal of the American Chemical Society, 134(10):4983-9 (2012), Kesselman, et al., Small, 8(7):1092-8 (2012)). This major limitation makes the maintenance of cells requiring adhesive ligands for viability and function difficult. Lutolf devised a microfluidic scheme to generate surface-modifiable synthetic microgels that does not utilize free radical polymerization, but neither bulk modification with bioactive molecules nor cell encapsulation was shown (Allazetta, et al., Biomacromolecules, 14(4):1122-31 (2013), Panda, et al., Lab Chip, 8(7):1056-61 (2008), Chung, et al., Applied Physics Letters, 91:041106 (2007)).
Microfluidic encapsulation of large clusters of cells, such as human islets, is more challenging than single cell encapsulation, because the larger particles tend to clog microfluidic channels. To minimize encapsulation volume while avoiding microfluidics altogether, investigators have explored conformal coating of islets (Blasi, et al., International Journal of Pharmaceutics, 440(2):141-7 (2013), Teramura, et al., Biomaterials, 34(11):2683-93 (2013)). Whereas conformal coating minimizes transplant volume, the immunoisolation potential of such thin polymer membranes remains unknown.
Thus there remains a need for improved, biocompatible methods of encapsulating cells and other biological agents in microgels.
It is therefore an object of the invention to provide a tunable, biocompatible platform for packaging cells and/or other biological agents, including, but not limited to, peptides, proteins, nucleic acids, and other biomolecules, into microgels.
It is a further object of the invention to provide a tunable, biocompatible platform for packaging multiple cells, for example cell clusters or islets, into a single microgel droplet.
It is another object of the invention to provide compositions including microgel encapsulated cells and other biological agents.
It is another object of the invention to provide methods of using microgel encapsulated cells and both biological agents.